Protein C-Factor VII Chimeras

ABSTRACT

Provided are chimeric Protein C-Factor VII proteins comprising a Gla domain from Protein C (PC), an EGF-1 domain from PC, an EGF-2 domain from Factor VII (FVII), and a protease domain from FVII.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 62/714,189, filed on Aug. 3, 2018, the entirecontents of which are hereby incorporated by reference.

BACKGROUND

Blood coagulation factor VII (FVII) is a glycoprotein that is found innormal human plasma. When vascular injury occurs, trace amounts of theactivated form of FVII (FVIIa) bind to an endothelial cell transmembranereceptor, tissue factor (TF), that becomes exposed at the site ofinjury. Under physiological conditions, TF-bound FVIIa (TF:FVIIa)rapidly activates blood coagulation Factor X (FX) leading toamplification of the intrinsic blood coagulation pathway for effectivefibrin clot formation and hemostasis. Under pathological conditions, TFbinding to FVIIa can lead to excessive fibrin clot formation andlife-threatening thrombosis.

Within the vasculature, FVII/FVIIa is known to bind to a transmembranereceptor, endothelial cell Protein C receptor (EPCR), the primaryreceptor for Protein C (PC) and its activated form (APC) (Fukudome etal., 1994). APC is a potent anticoagulant enzyme that proteolyticallyinactivates blood coagulation factors Va and VIIIa, therebydown-regulating thrombin generation and fibrin clot formation (Kisiel,1979). Pharmacologically administered recombinant FVIIa (rFVIIa)competitively inhibits PC binding to EPCR and PC activation bythrombin:thrombomodulin (Ghosh et al., 2007) to reduce the anticoagulanteffects of APC and thereby contribute to the hemostatic effectiveness ofrFVIIa (Keshava et al., 2017).

Severe hemophilia A and B are characterized by spontaneous bleedingepisodes, resulting in an overall mortality rate six times greater thanthe unaffected population. Because hemophilia is caused by a deficiencyof critical coagulation factors, standard treatments rely on replacingthe missing factor with recombinant or plasma-derived protein.Unfortunately, up to 33% of severe hemophilia patients developneutralizing alloantibodies against these replacement factors, renderingthem ineffective. Once the development of alloantibodies occurs, thetreatment options for acute bleeding are extremely limited. Currently,the preferred treatment option involves the use of rFVIIa to bypass themissing factors by binding to platelets and activating Factor X directlyon the platelet surface. However, the use of rFVIIa requires frequenthigh dosing at significant cost, and is limited by an inconsistentresponse with pronounced interpatient variability.

In 1998, it was speculated that rFVIIa might also be useful as a“universal” hemostatic agent in patients without blood coagulationdefects, who were suffering from uncontrolled bleeding for reasons otherthan hemophilia, e.g., bleeding related to surgery or trauma (Hedner,1998). In the years that have followed, there is evidence that rFVIIamay be effective in minimizing blood loss in a variety of clinicalsettings; however, the known risk of TF-driven thrombosis has been astrong deterrent for early intervention, and has led to the use oftreatment regimens with limited dosages and frequency of administration.Metanalyses that have generally not considered these constraints haveconcluded that the use of rFVIIa to control bleeding is ineffective andunsafe in non-hemophilia patients (Yank et al., 2011).

There is a need to develop FVII variants that display reduced tissuefactor-dependent thrombogenicity. Previous attempts to improve rFVIIatherapy have failed, in part, because the mechanism of platelet-rFVIIabinding is not well understood.

SUMMARY OF THE INVENTION

Some of the main aspects of the present invention are summarized below.Additional aspects are described in the Detailed Description of theInvention, Examples, Drawings, and Claims sections of this disclosure.The description in each section of this disclosure is intended to beread in conjunction with the other sections. Furthermore, the variousembodiments described in each section of this disclosure can be combinedin various different ways, and all such combinations are intended tofall within the scope of the present invention.

The therapeutic mechanism of action of rFVIIa has been shown to involveactivated platelet binding, as opposed to TF binding (Monroe et al.,1997). We have recently discovered that human platelets express EPCR,suggesting that modulation of EPCR binding could be utilized to enhancethe hemostatic efficacy of FVIIa variants (Fager et al. 2018). Thepresent disclosure provides procoagulant PC-FVII chimeras that bind toEPCR on platelets and on endothelial cells, and displayanti-inflammatory properties and endothelial cell barrier stabilizationproperties. In addition, the PC-FVII chimeras display no detectablebinding to TF, leading to reduced TF-dependent thrombogenicity comparedto wild-type or recombinant FVII.

In particular, the invention provides a chimeric PC-FVII proteincomprising a Gla domain of PC, an EGF-1 domain of PC, an EGF-2 domain ofFVII, and a protease domain of FVII. An exemplary chimeric PC-FVIIprotein of the invention has the amino acid sequence set forth in SEQ IDNO: 6 and is referred to herein as PC_(gla-egf1)FVIIa. The activatedform of the chimeric PC-FVII proteins of the invention is a heterodimer.

Accordingly, in one form, the chimeric PC-FVII protein is comprised of asingle amino acid chain. In another form, chimeric PC-FVII proteincomprises (i) a light chain comprising a Gla domain from PC, anEGF-1domain from PC, and an EGF-2 domain from FVII; and (ii) a heavychain comprising a protease domain from FVII. The light chain and theheavy chain are linked to one another, preferably by a disulfide bond.Other linkages can include, for example, salt bridges, ionic bonds,peptide linkers, and/or lactam bridges.

In one embodiment, the Gla domain comprises the amino acid sequence setforth in SEQ ID NO: 9. In one embodiment, the EGF-1 domain comprises theamino acid sequence set forth in SEQ ID NO: 12. In one embodiment, theEGF-2 domain comprises the amino acid sequence set forth in SEQ ID NO:11. In one embodiment, the protease domain comprises the amino acidsequence set forth in SEQ ID NO: 14. In one embodiment, the proteasedomain comprises the amino acid sequence set forth in SEQ ID NO: 15.

In a particular embodiment, the invention provides a chimeric PC-FVIIprotein comprising the amino acid sequence set forth in SEQ ID NO: 6.

In some embodiments, the chimeric PC-FVII protein of the inventioncomprises a propeptide sequence, wherein the propeptide sequence iscapable of binding vitamin K-dependent γ-glutamyl carboxylase. In someembodiments, the chimeric PC-FVII protein further comprises anendoplasmic reticulum translocalization signal peptide.

In a particular embodiment, the invention provides a chimeric PC-FVIIprotein comprising the amino acid sequence set forth in SEQ ID NO: 5.

A further aspect of the invention provides a composition comprising achimeric PC-FVII protein of the invention. In one embodiment, thecomposition is a pharmaceutical composition.

An additional aspect of the invention provides a kit comprising achimeric PC-FVII protein or a composition of the invention. In oneembodiment, the composition is contained in a pre-filled syringe.

The invention also provides a nucleic acid encoding a chimeric PC-FVIIprotein of the invention. In one embodiment, the nucleic acid comprisesthe nucleotide sequence set forth in SEQ ID NO: 7.

Also provided are chimeric PC-FVII proteins and compositions of theinvention for use in activating Factor X (FX), and a method ofactivating FX. The method comprises contacting FX with an activatedchimeric PC-FVII protein of the invention, wherein the chimeric PC-FVIIprotein cleaves FX, thereby producing activated Factor X (FXa). In oneembodiment, the method is performed in the absence of TF. In oneembodiment the method is performed in blood or plasma. In a particularembodiment, activating FX by contacting it with a chimeric PC-FVIIprotein results in increased thrombin concentration in the blood orplasma, compared to contacting FX with wild-type or recombinant FVIIa.In certain embodiments, the methods and uses of the invention areperformed in vitro or ex vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the structure of a representative chimera of theinvention.

The PC_(gla-egf1)FVIIa chimera contains the protease and EGF-2 domainsof Factor VIIa (FVIIa) with the EGF-1 and Gla domains of Protein C (PC).FIG. 1A shows a ribbon diagram of the predicted structure ofPC_(gla-egf1)FVIIa, based on the crystal structures of FVIIa and PC.FIG. 1B shows the unactivated or zymogen form of PC_(gla-egf1)FVII(PC-FVII), which is in the form of a single-chain polypeptide. Thesingle-chain zymogen is converted into a two-chain, disulfidebond-linked, activated form of PC-FVII (PC-FVIIa) by proteolyticcleavage of the Arg159-Ile160 peptide bond (FIG. 1C).

FIG. 2 shows screening of the initial chimera clones. Media from fiveseparate PC_(gla-egf1)FVIIa clones (DB1-5) was assayed for activity bymonitoring the cleavage of a FVIIa chromogenic substrate. Activity wascompared to media from a cell-free well (blank).

FIG. 3 shows gel electrophoresis of purified PC_(gla-egf1)FVIIa. Thechimera was isolated from conditioned media of clones expressingsignificant FVIIa activity. The starting media (lane 1) and eluates fromtwo separate purifications A (lanes 3-4) and B (lanes 5-6) weresubjected to SDS-PAGE under reducing (R) and non-reducing (NR)conditions as shown. Full length (FL) chimera is seen for allpurifications. The samples from purification A are partially activatedas evidenced by the presence of both Heavy Chain (HC) and Light Chain(LC) portions under reducing conditions. Molecular weight markers (MW)are shown in lane 2.

FIG. 4 shows PC_(gla-egf1)FVIIa autoactivation. PurifiedPC_(gla-egf1)FVIIa (2 μM) was incubated for varying amounts of time inthe absence of calcium (▪) prior to determining the amount ofautoactivated chimera as evidenced by its ability to cleave a FVIIasubstrate. The amount of autoactivation was compared to chimeraincubated in the presence of calcium (5 mM) either with (▴) or without(●) phospholipid (100 μM). The rate of autoactivation was also comparedto the rate of activation by 20 nM FXa in the presence of both calciumand phospholipid (▾). Rates are expressed as absorbance change perminute (mOD/min).

FIG. 5 shows that rFVIIa, PC_(gla-egf1)FVIIa, and FIX_(gla-egf1)FVIIahave similar rates of FVIIa substrate cleavage. Antithrombin was usedfor active-site titration to determine the relative proteinconcentration and activity of each molecule. 200 nM of either rFVIIa,PC_(gla-egf1)FVIIa, or FIX_(gla-egf1)FVIIa was incubated with 5 U/ml ofheparin and varying amounts of antithrombin (0-800 nM) prior to adding achromogenic substrate for FVIIa (Pefachrome VIIa, 500 FVIIa activity wasassessed by continuously monitoring cleavage of the chromogenicsubstrate. Data shown are linear rates of FVIIa substrate cleavage pernM of enzyme.

FIGS. 6A-6B show that Tissue Factor (TF)-independent activity ofPC_(gla-egf1)FVIIa is increased compared to FIX_(gla-egf1)FVIIa andrFVIIa. FIG. 6A shows phospholipid vesicles (40 μM) incubated with 20 nMrFVIIa (●), PC_(gla-egf1)FVIIa (▪), or FIX_(gla-egf1)FVIIa (▴). Plasmalevels (135 nM) of FX were added and FXa generation was assessed bycontinuously monitoring cleavage of a chromogenic substrate (PefachromeXa). Data shown are actual FXa generation rates as a function ofabsorbance at 405 nm over time. FIG. 6B shows linear rates of FXageneration (mOD/min), calculated from the data shown in FIG. 6A.

FIGS. 7A-7B show that PC_(gla-egf1)FVIIa has substantially reducedaffinity for TF compared to rFVIIa. FIG. 7A shows TF-dependent activity,assessed by incubating varying amounts of rFVIIa (0-2000 pM) (●), 2000pM PC_(gla-egf1)FVIIa (▪), 2000 pM FIX_(gla-egf1)FVIIa (♦), or vehiclecontrol (▴) with 1 nM TF (Innovin). Plasma levels (135 nM) of FX wereadded and FXa generation was assessed by continuously monitoringcleavage of a chromogenic substrate (Pefachrome Xa). Data shown areactual FXa generation rates as a function of absorbance at 405 nm overtime. FIG. 7B shows linear rates of FXa generation (mOD/min), calculatedfor each molecule from the data shown in FIG. 7A. The lack of FXactivation, even at 100-fold higher concentration than that of rFVIIa,confirms that the interaction between TF and these chimeras issignificantly reduced.

FIGS. 8A-8D show that PC_(gla-egf1)FVIIa and rFVIIa are sensitive tolipid concentration. Thrombin generation was assessed using a calibratedautomated thrombography (CAT) assay. Hemophilia A (FVIII deficient)plasma was incubated either with (FIG. 8A) or without (FIG. 8B) 2 U/mLFVIII, followed by the addition of 1 pM TF and either 4 μM (▪), 30 μM(♦), or 300 μM (▴) phospholipid vesicles in the presence of afluorogenic thrombin substrate (432 μM). Thrombin generation wasassessed by continuously monitoring the resulting change in fluorescenceintensity as a result of substrate cleavage. Similar experiments wereperformed in Hemophilia A plasma following the addition of equalconcentrations (20 μg/mL) of either rFVIIa (FIG. 8C) orPC_(gla-egf1)FVIIa (FIG. 8D). Data shown are actual thrombin generationrates as a function of the change in fluorescence intensity over time.

FIG. 9 shows that PC_(gla-egf1)FVIIa facilitates increasedTF-independent thrombin generation compared to rFVIIa. Thrombingeneration was assessed using a modified CAT assay in the absence of TF.Hemophilia A (HA) plasma was incubated with 20 μg/mL ofPC_(gla-egf1)FVIIa (▪), rFVIIa (▴), or buffer control (♦), followed bythe addition of varying amounts of phospholipid vesicles (3-30 μM) inthe presence of a fluorogenic thrombin substrate (432 μM). Thrombingeneration was assessed by continuously monitoring the resulting changein fluorescence intensity as a result of substrate cleavage. Data shownare actual thrombin generation rates as a function of the change influorescence intensity over time.

FIGS. 10A-10F show the amino acid sequences of human FVII zymogen (FIG.10A; SEQ ID NO: 1); human FVII (FIG. 10B; SEQ ID NO: 2); human PCzymogen (FIG. 10C; SEQ ID NO: 3); human PC (FIG. 10D; SEQ ID NO: 4); aPC-FVII chimeric zymogen of the invention (FIG. 10E; SEQ ID NO: 5); aPC-FVII chimera of the invention, PC_(gla-egf1)FVIIa (FIG. 10F; SEQ IDNO: 6); and a cDNA sequence encoding the PC_(gla-egf1)FVII zymogen (FIG.10G; SEQ ID NO: 7), including cloning sites. The bolded residues in FIG.10E and FIG. 10F indicate amino acid sequences from PC. The italicizedresidues in FIG. 10E and FIG. 10F indicate amino acid sequences fromFVII. The bolded, italicized, underlined residues in FIG. 10E and FIG.10F indicate the FVII activation site; cleavage of the peptide bondbetween these two residues produces a light chain and heavy chain, whichremain linked by a disulfide bond, activating the molecule. The bolded,underlined nucleotides in FIG. 10G indicate the start and stop codons.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of pharmaceutics, formulationscience, protein chemistry, cell biology, cell culture, molecularbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art.

In order that the present invention can be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the disclosure. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionis related.

Any headings provided herein are not limitations of the various aspectsor embodiments of the invention, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

All references cited in this disclosure are hereby incorporated byreference in their entireties. In addition, any manufacturers'instructions or catalogues for any products cited or mentioned hereinare incorporated by reference. Documents incorporated by reference intothis text, or any teachings therein, can be used in the practice of thepresent invention. Documents incorporated by reference into this textare not admitted to be prior art.

I. Definitions

The phraseology or terminology in this disclosure is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents, unless the contextclearly dictates otherwise. The terms “a” (or “an”) as well as the terms“one or more” and “at least one” can be used interchangeably.

Furthermore, “and/or” is to be taken as specific disclosure of each ofthe two specified features or components with or without the other.Thus, the term “and/or” as used in a phrase such as “A and/or B” isintended to include A and B, A or B, A (alone), and B (alone). Likewise,the term “and/or” as used in a phrase such as “A, B, and/or C” isintended to include A, B, and C; A, B, or C; A or B; A or C; B or C; Aand B; A and C; B and C; A (alone); B (alone); and C (alone).

Wherever embodiments are described with the language “comprising,”otherwise analogous embodiments described in terms of “consisting of”and/or “consisting essentially of” are included.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range, and any individual value provided herein canserve as an endpoint for a range that includes other individual valuesprovided herein. For example, a set of values such as 1, 2, 3, 8, 9, and10 is also a disclosure of a range of numbers from 1-10, from 1-8, from3-9, and so forth. Likewise, a disclosed range is a disclosure of eachindividual value encompassed by the range. For example, a stated rangeof 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, can comprise modifiedamino acids, and can be interrupted by non-amino acids. Except whereindicated otherwise, e.g., for the abbreviations for the uncommon orunnatural amino acids set forth herein, the three-letter and one-letterabbreviations, as used in the art, are used herein to represent aminoacid residues. Except where specifically indicated, peptides areindicated with the N-terminus of the left and the sequence is writtenfrom the N-terminus to the C-terminus.

Polypeptides, peptides, and proteins can comprise natural or syntheticpost-translational modifications, for example, disulfide bonds, lactambridges, carboxylation, hydroxylation, glycosylation, lipidation,alkylation, acetylation, acylation, amidation, phosphorylation, or othermanipulations or modification, such as conjugation with a labelingcomponent or addition of a protecting group. Also included are, forexample, polypeptides containing one or more analogs of an amino acid(including, for example, amino-isobutyric acid (Aib), unnatural aminoacids, such as naphthylalanine (Nal), etc.), as well as othermodifications known in the art. In certain embodiments, the polypeptidescan occur as single chains, covalent dimers, or non-covalent associatedchains.

A “chimera” or “chimeric” molecule is one comprising structural featuresof more than one reference molecule. In the context of the presentinvention, a chimeric protein comprises an amino acid sequence from afirst polypeptide and an amino acid sequence from a second polypeptide.The amino acid sequences can be linked covalently, such as, for example,by peptide bonds or disulfide bonds. The amino acid sequences can becontiguous, i.e., directly fused, or can comprise a linker, such as apeptide linker, between the amino acid sequence from a first polypeptideand the amino acid sequence of a second polypeptide.

The term “coagulation factor” refers to a protein involved in thecoagulation cascade, in either its activated or zymogen form.Coagulation factors include serine proteases, such as Factor VII, FactorIX, Factor X, Factor XI, Factor XII, prothrombin, and Protein C;glycoproteins, such as Factor V, Factor VIII, and protein S; andtransglutaminases, such as Factor XIII.

The term “variant” refers to a peptide having one or more amino acidsubstitutions, deletions, and/or insertions compared to a referencesequence. Deletions and insertions can be internal and/or at one or moretermini.

The term “conservative substitution” as used herein denotes that one ormore amino acids are replaced by another, biologically similar residue.Examples include substitution of amino acid residues with similarcharacteristics, e.g., small amino acids, acidic amino acids, polaramino acids, basic amino acids, hydrophobic amino acids, and aromaticamino acids. For further information concerning phenotypically silentsubstitutions in peptides and proteins, see, for example, Bowie et. al.,Science 247:1306-1310 (1990). In Table I, conservative substitutions ofamino acids are grouped by physicochemical properties; I: neutral and/orhydrophilic, II: acids and amides, III: basic, IV: hydrophobic, V:aromatic, bulky amino acids.

TABLE I I II III IV V A N H M F S D R L Y T E K I W P Q V G C

In Table II, conservative substitutions of amino acids are grouped byphysicochemical properties; VI: neutral or hydrophobic, VII: acidic,VIII: basic, IX: polar, X: aromatic.

TABLE II VI VII VIII IX X A D H M F L E R S Y I K T W V N H P Q G C

Methods of identifying conservative nucleotide and amino acidsubstitutions which do not affect protein function are well-known in theart (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashiet al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc.Natl. Acad. Sci. U.S.A. 94:412-417 (1997)).

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or peptides, refers to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity can be measured using sequencecomparison software or algorithms, or by visual inspection. Variousalgorithms and software are known in the art that can be used to obtainalignments of amino acid or nucleotide sequences.

One such non-limiting example of a sequence alignment algorithm isdescribed in Karlin et al., Proc. Natl. Acad. Sci., 87:2264-2268 (1990),as modified in Karlin et al., Proc. Natl. Acad. Sci., 90:5873-5877(1993), and incorporated into the NBLAST and XBLAST programs (Altschulet al., Nucleic Acids Res., 25:3389-3402 (1991)). In certainembodiments, Gapped BLAST can be used as described in Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul etal., Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2(Genentech, South San Francisco, Calif.) or Megalign (DNASTAR) areadditional publicly available software programs that can be used toalign sequences. In certain embodiments, the percent identity betweentwo nucleotide sequences is determined using the GAP program in the GCGsoftware package (e.g., using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). Incertain alternative embodiments, the GAP program in the GCG softwarepackage, which incorporates the algorithm of Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)), can be used to determine the percentidentity between two amino acid sequences (e.g., using either a BLOSUM62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certainembodiments, the percent identity between nucleotide or amino acidsequences is determined using the algorithm of Myers and Miller (CABIOS4:11-17 (1989)). For example, the percent identity can be determinedusing the ALIGN program (version 2.0) and using a PAM120 with residuetable, a gap length penalty of 12 and a gap penalty of 4. One skilled inthe art can determine appropriate parameters for maximal alignment byparticular alignment software. In certain embodiments, the defaultparameters of the alignment software are used. Other resources forcalculating identity include methods described in ComputationalMolecular Biology (Lesk ed., 1988); Biocomputing: Informatics and GenomeProjects (Smith ed., 1993); Computer Analysis of Sequence Data, Part 1(Griffin and Griffin eds., 1994); Sequence Analysis in Molecular Biology(G. von Heinje, 1987); Sequence Analysis Primer (Gribskov et al. eds.,1991); and Carillo et al., SIAM J. Applied Math., 48:1073 (1988).

A “polynucleotide,” as used herein can include one or more “nucleicacids,” “nucleic acid molecules,” or “nucleic acid sequences,” andrefers to a polymer of nucleotides of any length, and includes DNA andRNA. The polynucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase. Apolynucleotide can comprise modified nucleotides, such as methylatednucleotides and their analogs. The preceding description applies to allpolynucleotides referred to herein, including RNA and DNA.

An “isolated” molecule is one that is in a form not found in nature,including those which have been purified.

A “label” is a detectable compound that can be conjugated directly orindirectly to a molecule, so as to generate a “labeled” molecule. Thelabel can be detectable on its own (e.g., radioisotope labels orfluorescent labels), or can be indirectly detected, for example, bycatalyzing chemical alteration of a substrate compound or compositionthat is detectable (e.g., an enzymatic label) or by other means ofindirect detection (e.g., biotinylation).

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a moleculeand its binding partner (e.g., a receptor and its ligand, an antibodyand its antigen, two monomers that form a dimer, etc.). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair. The affinity of a molecule X for its partner Y cangenerally be represented by the dissociation constant (K_(D)). Affinitycan be measured by common methods known in the art, including thosedescribed herein. Low-affinity binding partners generally bind slowlyand tend to dissociate readily, whereas high-affinity binding partnersgenerally bind faster and tend to remain bound longer.

The affinity or avidity of a molecule for its binding partner can bedetermined experimentally using any suitable method known in the art,e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), orradioimmunoassay (MA), or kinetics (e.g., KINEXA® or BIACORE™ or OCTET®analysis). Direct binding assays as well as competitive binding assayformats can be readily employed. (See, e.g., Berzofsky et al.,“Antibody-Antigen Interactions,” in Fundamental Immunology, Paul, W. E.,ed., Raven Press: New York, N.Y. (1984); Kuby, Immunology, W. H. Freemanand Company: New York, N.Y. (1992)). The measured affinity of aparticular binding pair interaction can vary if measured under differentconditions (e.g., salt concentration, pH, temperature). Thus,measurements of affinity and other binding parameters (e.g., K_(D) orKd, K_(on), K_(off)) are made with standardized solutions of bindingpartners and a standardized buffer, as known in the art.

An “active agent” is an ingredient that is intended to furnishbiological activity. The active agent can be in association with one ormore other ingredients.

An “effective amount” of an active agent is an amount sufficient tocarry out a specifically stated purpose.

The term “pharmaceutical composition” refers to a preparation that is insuch form as to permit the biological activity of the active ingredientto be effective and which contains no additional components that areunacceptably toxic to a subject to which the composition would beadministered. Such composition can be sterile and can comprise apharmaceutically acceptable carrier, such as physiological saline.Suitable pharmaceutical compositions can comprise one or more of abuffer, a surfactant, a stabilizing agent, a preservative, and/or otherconventional solubilizing or dispersing agents.

The terms “inhibit,” “block,” and “suppress” are used interchangeablyand refer to any statistically significant decrease in occurrence oractivity, including full blocking of the occurrence or activity. Forexample, “inhibition” can refer to a decrease of about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence. An“inhibitor” is a molecule, factor, or substance that produces astatistically significant decrease in the occurrence or activity of aprocess, pathway, or molecule.

II. Protein C-Factor VII Chimeras

Blood coagulation factors activated Factor VII (FVIIa) and ActivatedProtein C (APC) are vitamin K-dependent, glycosylated, heterodimericserine proteases, each derived from a zymogen precursor. The canonicalamino acid sequence of the human FVII zymogen is set forth in GenBankAccession No. AAA88040 and in FIG. 10A; that of human PC is set forth inGenBank Accession No. P04070 and in FIG. 10C. A number of naturalvariants of each protein have been identified (see, e.g., UniProtKBEntry P08709; UniProtKB Entry P04070). Sequences from such variants aresuitable for use in the PC-FVII chimeras of the invention.

Mature FVIIa contains 406 amino acids and results from a singleproteolytic cleavage between amino acids R152 and 1153 of FVII; theN-terminal signal peptide and propeptide are also cleaved (e.g., SEQ IDNO: 2; FIG. 10B). Following cleavage of its signal peptide andpropeptide sequences, PC contains 419 amino acids (e.g., SEQ ID NO: 4;FIG. 10D).

FVIIa and PC share a similar molecular structure with other coagulationserine proteases (e.g., Factor IXa and Factor Xa), each having a lightchain and a heavy chain. The light chain is comprised of an aminoterminal gamma-carboxyglutamic acid (Gla) domain and two epidermalgrowth factor (EGF)-like domains, while the heavy chain is comprised ofa protease domain (see Lazarus et al., 2004).

A “Gla domain” is an amino acid sequence from a coagulation factor,which amino acid sequence binds directly to phospholipid membranes. TheGla domains of FVII/FVIIa and PC can also bind to phospholipid membranesvia the transmembrane endothelial cell Protein C receptor (EPCR) (seeGhosh et al., 2007). PC binding to EPCR promotes PC activation by thethrombin:thrombomodulin complex. The Gla domain of FVII/FVIIa cancomprise, for example, amino acids from about position 1 to aboutposition 45 of SEQ ID NO: 2 (SEQ ID NO: 8). The Gla domain of PC cancomprise, for example amino acids from about position 1 to aboutposition 46 of SEQ ID NO: 4 (SEQ ID NO: 9). The Gla domain can includepost-translational modifications, for example, carboxylation, such asγ-carboxylation, and/or hydroxylation, such as β-hydroxylation.

An “EGF domain” is a cysteine-rich amino acid sequence, typically about30-45 residues in length, found originally in epidermal growth factor. Arange of proteins involved in cell signaling and in the coagulationcascade contain EGF domains. The EGF domains of FVIIa and PC areinvolved in cofactor recognition. For example, the EGF-1 domain of FVIIaplays a critical role in its affinity for TF. The EGF-1 domain of PC, onthe other hand, is not known to have an affinity for TF. The EGF-1domain of FVII/FVIIa can comprise, for example, amino acids from aboutposition 46 to about position 82 of SEQ ID NO: 2 (SEQ ID NO: 10). TheEGF-2 domain of FVII/FVIIa can comprise, for example, amino acids fromabout position 87 to about position 128 of SEQ ID NO: 2 (SEQ ID NO: 11).The EGF-1 domain of PC can comprise, for example, amino acids from aboutposition 55 to about position 90 of SEQ ID NO: 4 (SEQ ID NO: 12). TheEGF-2 domain of PC can comprise, for example, amino acids from aboutposition 94 to about position 134 of SEQ ID NO: 4 (SEQ ID NO: 13). EachEGF domain can include post-translational modifications, for example,hydroxylation, such as β-hydroxylation, and/or glycosylation, such asN-glycosylation and/or O-glycosylation and/or fucosylation.

The protease domain of FVIIa is responsible for its cleavage/activationof Factor IX and Factor X. Activated Protein C (APC) proteolyticallyinactivates Factor Va and Factor VIIIa to downregulate the process ofblood coagulation, which has direct anti-coagulant, anti-hemostaticand/or anti-thrombotic effects. FVIIa binding to EPCR can displace boundPC to competitively inhibit PC activation, which could have indirectpro-coagulant, pro-hemostatic and/or pro-thrombotic effects. Theprotease domain of FVII/FVIIa can comprise, for example, amino acidsfrom about position 153 to about position 392 of SEQ ID NO: 2 (SEQ IDNO: 14), or from about position 153 to about position 406 of SEQ ID NO:2 (SEQ ID NO: 15). The protease domain of PC can comprise, for example,amino acids from about position 169 to about position 408 of SEQ ID NO:4 (SEQ ID NO: 16). The protease domain can include post-translationalmodifications, for example, phosphorylation and/or glycosylation, suchas N-glycosylation.

The present inventors have discovered that chimeric PC-FVIIa comprisingGla and EGF1 domains from PC and EGF2 and protease domains from FVIIahas the membrane-binding and receptor-targeting properties of PC and theenzymatic activity of FVIIa, without the Tissue Factor (TF) binding ofFVIIa. The interaction between pharmacologically administered rFVIIa andTF is an established mechanism for thrombotic complications that haveoccurred in a clinical context. In their activated form, the PC-FVIIchimeras of the invention can bind to EPCR on endothelial cells, whichcan cleave PAR1 and initiate intracellular signaling, leading toanti-inflammatory effects and preservation of the barrier function ofendothelium.

Non-limiting examples of amino acid sequences that can be used in theinvention are set forth in Table 1.

TABLE 1 Domain SEQ ID NO Amino Acid Sequence FVII Gla  8ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYS PC Gla  9ANSFLEELRHSSLERECIEEICDFEEAKEIFQNVDDTLAFWSKHVD FVII EGF-1 10DGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCE FVII EGF-2 11DQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCT PC EGF-1 12LEHPCASLCCGHGTCIDGIGSFSCDCRSGWEGRFCQ PC-EGF-2 13SFLNCSLDNGGCTHYCLEEVGWRRCSCAPGYKLGDDLLQCH FVII Protease 14IVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTR VSQYIEWLQKLMR FVII Protease15 IVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTR VSQYIEWLQKLMRSEPRPGVLLRAPFPPC Protease 16 LIDGKMTRRGDSPWQVVLLDSKKKLACGAVLIHPSWVLTAAHCMDESKKLLVRLGEYDLRRWEKWELDLDIKEVFVHPNYSKSTTDNDIALLHLAQPATLSQTIVPICLPDSGLAERELNQAGQETLVTGWGYHSSREKEAKRNRTFVLNFIKIPVVPHNECSEVMSNMVSENMLCAGILGDRQDACEGDSGGPMVASFHGTWFLVGLVSWGEGCGLLHNYGVYT KVSRYLDWIHGHIR

PC-FVII chimeras of the invention can include domains comprising more,fewer, or conservatively substituted amino acids compared with thosesequences listed in Table 1, provided that the basic function of thedomain is preserved. The chimeras can comprise solely wild-type FVII andPC amino acid sequences. Alternatively, the chimeras can compriseheterologous sequences (i.e., from neither wild-type FVII nor wild-typePC), for example, the chimeras can comprise one or more heterologouslinkers between domains. The amino acid sequence of an exemplary PC-FVIIchimera is shown in FIG. 10F.

PC-FVII chimeras of the invention can comprise a prepropeptide. Theprepropeptide is typically comprised of a signal peptide, which directslocalization of the molecule to the lumen of the endoplasmic reticulum,and a propeptide, which binds vitamin K-dependent γ-glutamylcarboxylase. The prepropeptide sequence can be from FVII, PC, or anothercoagulation factor, preferably from another vitamin K-dependent protein,for example, prothrombin, FIX, FX, Protein S, or Protein Z. The signalpeptide and propeptide can be from the same or different proteins. Suchsignal and propeptide sequences are highly conserved and are known inthe art (Kriegler et al., 2018; Stanley et al., 1999; Pan et al., 1985).The amino acid sequence of an exemplary PC-FVII chimera comprising aprepropeptide is shown in FIG. 10E.

PC-FVII chimeras of the invention can optionally include one or moreepitope and/or affinity tags, such as for purification or detection.Non-limiting examples of such tags include FLAG, HA, His, Myc, GST, andthe like. PC-FVII chimeras of the invention can optionally include oneor more labels.

In certain aspects, the invention provides a composition, e.g., apharmaceutical composition, comprising a PC-FVII chimera of theinvention, optionally further comprising one or more carriers, diluents,excipients, or other additives.

Also within the scope of the invention are kits comprising the PC-FVIIchimeras and compositions as provided herein and, optionally,instructions for use. In one embodiment, the kit comprises a syringe.The syringe can be pre-filled with a composition comprising a PC-FVIIchimera of the invention. The kit can further contain at least oneadditional reagent, and/or one or more additional active agents. Kitstypically include a label indicating the intended use of the contents ofthe kit. In this context, the term “label” includes any writing orrecorded material supplied on or with the kit, or that otherwiseaccompanies the kit.

The PC-FVII chimeras can be used in various contexts, for example, as amodel for studying coagulation, EPCR-mediated binding, and the effectsof platelet vs. endothelial membrane microenvironment. PC-FVII chimeraactivity and function can be measured by known assays, including theassays described herein.

III. Methods of Preparation

PC-FVII chimeras of the invention can be chemically synthesized or canbe expressed using recombinant methods. Synthesis or expression mayoccur as fragments of the protein which are subsequently combined eitherchemically or enzymatically.

Accordingly, also provided are nucleic acid molecules encoding PC-FVIIchimeras of the invention. Nucleic acid molecules of the invention canbe designed based on the amino acid sequence of the desired PC-FVIIchimera and selection of those codons that are favored in the host cellin which the recombinant PC-FVII chimera will be produced. Standardmethods can be applied to synthesize a nucleic acid molecule encoding aPC-FVII chimera of interest. An exemplary nucleotide sequence encoding aPC-FVII chimera of the invention is shown in FIG. 10G.

Once prepared, the nucleic acid encoding a particular PC-FVII chimeracan be inserted into an expression vector and operably linked to anexpression control sequence appropriate for expression of the peptide ina desired host. In order to obtain high expression levels of the PC-FVIIchimera, the nucleic acid can be operably linked to or associated withtranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

A wide variety of expression host/vector combinations can be employed.Useful expression vectors for eukaryotic hosts include, for example,vectors comprising expression control sequences from SV40, bovinepapilloma virus, adenovirus, and cytomegalovirus. Useful expressionvectors for bacterial hosts include known bacterial plasmids, such asplasmids from E. coli, including pCR1, pBR322, pMB9 and theirderivatives, wider host range plasmids, such as M13, and filamentoussingle-stranded DNA phages.

Suitable host cells include prokaryotes, yeast, insect, or highereukaryotic cells under the control of appropriate promoters. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells can be established or cell lines ofmammalian origin, examples of which include Pichia pastoris, HEK293cells, COS-7 cells, L cells, C127 cells, 3T3 cells, Chinese hamsterovary (CHO) cells, HeLa cells, and BHK cells. Cell-free translationsystems can also be employed. Helper enzymes, such as vitaminK-dependent carboxylase (VKGC), vitamin K epoxide reductase (VKOR),and/or PACE/furin can be used to increase the levels of functionalvitamin K-dependent protein expression.

DNA sequences for use in producing PC-FVII chimeras of the inventionwill typically encode a prepropeptide at the N-terminus to obtain properpost-translational processing and secretion from the host cell. As willbe appreciate by those skilled in the art, additional modifications,such as amino acid additions, deletions, and substitutions, can be madein the amino acid sequence of the PC-FVII chimeras described herein,provided that those modifications do not significantly affect theprotein's function.

EXAMPLES

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples. It will be apparent tothose skilled in the art that many modifications, both to materials andmethods, can be practiced without departing from the scope of thepresent disclosure. A number of variants to reference peptide ST-3 weremade and examined, as described below.

Example 1. Expression and Purification of PC_(gla-egf1)FVIIa

We designed a chimeric cDNA encoding residues 1-91 (Gla and EGF1domains) of human PC linked to residues 85-406 (EGF2 and Proteasedomains) of human FVIIa (PC_(gla-egf1)FVIIa) (FIG. 1). The cDNA encodingPC_(gla-egf1)FVIIa was synthesized and cloned into a pCDNA3.1(+) pCMVexpression vector (OriGene). The vector was then stably transfected intohuman embryonic kidney (HEK293) cells (ATCC). Colonies were cloned intoindividual wells of a 24-well plate, and initial clones of interest wereselected and screened (FIG. 2) for expression of the construct bymonitoring cleavage of a chromogenic FVIIa substrate (Pentapharm).Briefly, conditioned media was collected and centrifuged to remove celldebris. The supernatant was concentrated using a Microcon-30 (MilliporeSigma) and activated by incubating with FXa (2 nM) and PS/41% PC/44% PEphospholipid vesicles (40 μM), in the presence of 3 mM CaCl₂ at RT for30 minutes. Following activation, Rivaroxaban (4.75 mM) was added toinhibit residual FXa activity. Samples were incubated at RT for 5 minprior to adding 1 mM Pefachrome FVIIa. FVIIa activity was subsequentlywas assessed by continuously monitoring the cleavage of the chromogenicsubstrate at 405 nm every 12 seconds for 1 hour.

Candidate clones expressing significant FVIIa activity were expanded forpurification of the chimera using sequential Q-Sepharose (GE Healthcare)and HiTrap S (GE Healthcare) columns eluted with a salt gradient asdescribed (Chang et al., 1995). Two separate purifications (A and B)were completed using conditioned media from two separate clones. Theeluates from each of the purification procedures were subjected toprotein electrophoresis using 10-15% Phast gels (Pharmacia Biotech). Theresulting bands are consistent with the expected molecular weight ofPC_(gla-egf1)FVIIa (FIG. 3). Protein concentration was also determinedusing a BCA assay (Pierce).

Example 2. PC_(gla-egf1)FVIIa is Capable of Autoactivation

During the initial screens for chimera activity, the conditioned mediafrom each candidate clone was purposefully activated by FXa as above toensure activation of the chimera prior to screening for activity.However, protein electrophoresis of the eluate from the firstpurification procedure (FIG. 3 Purification A) showed that the chimerawas isolated in a partially activated form even in the absence ofpre-treatment with FXa. This same partial activation was not seen in theeluate from the second purification. Since FVIIa is capable ofautoactivation, we evaluated the ability of the PC_(gla-egf1)FVIIa toautoactivate as well. For these experiments PC_(gla-egf1)FVIIa (2 μM)from the second purification was incubated with or without FXa (20 nM),CaCl₂ (5 mM), or phospholipid vesicles (100 μM). Following varyingincubation periods (0-35 min), residual FXa and CaCl₂ were inhibited byadding Rivaroxaban (50 nM) and EDTA (5 mM). Pefachrome FVIIa (500 μm)was added and FVIIa activity was assessed by continuously monitoring thecleavage of the chromogenic substrate at 405 nm every 12 seconds for 30min.

As shown in FIG. 4, autoactivation of the chimera occurs in the presenceof calcium and phospholipid. However, autoactivation is significantlyimpaired in either the absence of calcium or in the presence of calciumwithout a phospholipid surface. As expected, the addition of FXa rapidlyaccelerates the activation of PC_(gla-egf1)FVIIa in the presence ofcalcium and phospholipid. Therefore, for experiments in which FXa cannotbe added, (particularly those requiring measurement of FXa activity),the chimera can readily be activated by incubating with calcium andphospholipid.

Example 3. PC_(gla-egf1)FVIIa is Able to Cleave a Chromogenic FVIIaSubstrate

FIX_(gla-egf1)FVIIa is a chimera having the Gla and EGF1 domains ofFactor IX and the EGF2 and protease domains of FVIIa (Chang et al.,1995). Antithrombin active-site titrations, as described (Grandoni etal., 2017), were performed to determine the relative proteinconcentration and enzymatic activity of PC_(gla-egf1)FVIIa andFIX_(gla-egf1)FVIIa in vitro. Since both contain the protease domain ofFVIIa, FIX_(gla-egf1)FVIIa could be used as a positive control.

For these experiments 200 nM of either PC_(gla-egf1)FVIIa,FIX_(gla-egf1)FVIIa, or rFVIIa was incubated with 2.5 U/ml heparin andvarying amounts (0-800 nM) of antithrombin for 14 hours at roomtemperature. Following incubation, 500 μM Pefachrome FVIIa was added andFVIIa activity was assessed by continuously monitoring the cleavage ofthis chromogenic substrate at 405 nm every 15 seconds for 10 min. Therate of substrate cleavage was then plotted against the originalconcentration of antithrombin in order to determine the concentration ofactive material in each sample.

As shown in FIG. 5, both PC_(gla-egf1)FVIIa and FIX_(gla-egf1)FVIIa areable to cleave the synthetic FVIIa substrate. In addition, similaramounts of active protein resulted in similar rates of FVIIa substratecleavage for all three molecules. This result confirms that the rate ofsubstrate cleavage is a good indication of the number of active sitesfor each chimera.

Example 4. PC_(gla-egf1)FVIIa has Increased Tissue Factor(TF)-Independent Activity

After confirming the ability of PC_(gla-egf1)FVIIa to cleave a syntheticpeptide substrate, we also wanted to determine its ability to cleave amore physiologic substrate. We therefore compared the activity ofPC_(gla-egf1)FVIIa to rFVIIa and to FIX_(gla-egf1)FVIIa on phospholipidvesicles (15% PS/41% PC/44% PE) by monitoring Factor X (FX) activationusing a chromogenic substrate for Factor Xa (FXa) as described (Fager etal., 2018; Hoffman et al., 2011). PC_(gla-egf1)FVIIa,FIX_(gla-egf1)FVIIa, or rFVIIa (20 nM) were incubated with FX (135 nM),Pefachrome FXa (500 μM), and 5 mM CaCl₂ in the presence of 40 μMphospholipid. FXa activity was assessed by monitoring cleavage of thechromogenic substrate at 405 nm.

As shown in FIG. 6, the rate of FX activation by PC_(gla-egf1)FVIIa wassignificantly higher than that of both rFVIIa (˜2.6 times) andFIX_(gla-egf1)FVIIa (˜2 times). These results suggest the potential forincreased hemostatic efficacy of PC_(gla-egf1)FVIIa compared to rFVIIaand FIX_(gla-egf1)FVIIa. This data demonstrates that EPCR is notrequired for PC-FVIIa binding to lipid membranes.

Example 5. PC_(gla-egf1)FVIIa has a Very Low Affinity for Tissue Factor(TF)

To determine the affinity of PC_(gla-egf1)FVIIa for TF, its TF-dependentactivity was measured by monitoring FX activation in an assay designedto detect weak TF binding. For these assays, 2000 pM PC_(gla-egf1)FVIIa,2000 pM FIX_(gla-egf1)FVIIa, or varying amounts of rFVIIa (0-2000 pM),were incubated with FX (135 nM), Pefachrome FXa (500 μM), and 5 mM CaCl₂in the presence of 1 nM human TF (Innovin, DADE/Behring) which wasdialyzed to remove excess calcium. FX activation was assessed bymonitoring cleavage of the chromogenic substrate at 405 nm.

As shown in FIG. 7, the activity of PC_(gla-egf1)FVIIa was identical tothe vehicle control indicating that the interaction betweenPC_(gla-egf1)FVIIa and TF is too weak to be measured. The very low levelof FX activation by PC_(gla-egf1)FVIIa, even at 10- to 100-fold higherconcentrations than that of rFVIIa, confirms that PC_(gla-egf1)FVIIa hasa substantially reduced affinity for TF.

This reduced affinity for TF represents a potential advantage forPC_(gla-egf1)FVIIa, as long-term exposure to high levels of FVIIa causespremature mortality due to thrombosis in TF-rich tissues (Aljamali etal., 2008) and with TF-bearing microparticles. Minimizing theinteraction with TF could lead to a decreased risk of thrombosis thatcan occur with rFVIIa (von Bruhl et al., 2012), particularly innon-hemophilia patients who are at higher risk for these complications(Yank et al., 2011).

Example 6. Hemostatic Activity of PC_(gla-egf1)FVIIa is Sensitive toLipid Concentration

Thrombin serves as the central regulator of hemostasis and thrombosis.The efficient generation of thrombin requires the assembly ofmultiprotein enzyme complexes that are assembled on an appropriatemembrane surface. We therefore used a calibrated automated thrombography(CAT) assay as described (Vavalle et al., 2014), to determine the effectof varied concentrations of phospholipid surfaces on the ability ofPC_(gla-egf1)FVIIa to facilitate thrombin generation. Briefly, FactorVIII deficient (hemophilia A) plasma was incubated with tissue factor (1pM) and either rFVIIa (20 μg/ml), PC_(gla-egf1)FVIIa (20 μg/ml), orFVIII (2 U/ml) in the presence of varied concentrations of phospholipidvesicles (4-300 μM) and 432 μM fluorogenic thrombin substrate(Z-Gly-Gly-Arg-AMC). The lag time, peak thrombin concentration, andendogenous thrombin potential were then determined from directmeasurement of the resulting change in fluorescence intensity over time.

As shown in FIG. 8A, thrombin generation in normal plasma is relativelyinsensitive to the available lipid concentration. However, the abilitiesof both rFVIIa (FIG. 8C) and PC_(gla-egf1)FVIIa (FIG. 8D) to facilitatethrombin generation in Hemophilia A (FVIII deficient) plasma are quitesensitive to the lipid concentration with maximum activity noted in the300 μM phospholipid range. Furthermore, while rFVIIa considerablyshortens the lag time (FIG. 8A vs. FIG. 8C), the PC_(gla-egf1)FVIIa doesnot, likely due to the binding of rFVIIa but not PC_(gla-egf1)FVIIa toTF.

Example 7. PC_(gla-egf1)FVIIa Facilitates Increased TF-IndependentThrombin Generation

Given the increased rate of TF-independent FX activation byPC_(gla-egf1)FVIIa as compared to rFVIIa, we wanted to confirm that thiswould translate into an increased ability to facilitate thrombingeneration. To do so, we used a modification of the CAT assay describedin Example 6 in which the TF was excluded. Briefly, Hemophilia A plasmawas incubated with either rFVIIa (20 μg/ml) or PC_(gla-egf1)FVIIa (20μg/ml), in the presence of phospholipid vesicles (3-30 μM) and afluorogenic thrombin substrate (432 μM). The lag time, peak thrombinconcentration, and endogenous thrombin potential were again determinedfrom direct measurement of the resulting change in fluorescenceintensity over time.

As shown in FIG. 9, in the absence of TF, no appreciable thrombingeneration was seen in hemophilia A plasma alone. However, the additionof either rFVIIa or PC_(gla-egf1)FVIIa resulted in significantlyincreased thrombin generation. Furthermore, the addition ofPC_(gla-egf1)FVIIa resulted in increased peak thrombin concentration andendogenous thrombin potential with a longer lag time compared to rFVIIain this experiment. The increase in TF-independent thrombin generationobserved in the in vitro thrombin generation (CAT) assay suggests thepotential for increased hemostatic efficacy of PC_(gla-egf1)FVIIacompared to rFVIIa. In addition, the lack of measurable TF-dependentactivity also suggests that PC_(gla-egf1)FVIIa has the potential fordecreased thrombogenicity compared to rFVIIa.

REFERENCES

-   Aljamali M N, et al. Long-term expression of murine activated factor    VII is safe, but elevated levels cause premature mortality. J. Clin.    Invest. 118:1825-1834 (2008).-   Chang J Y, et al. The roles of factor VII's structural domains in    tissue factor binding. Biochemistry 34:12227-12232 (1995).-   Fager A M, et al. Human platelets express endothelial protein C    receptor, which can be utilized to enhance localization of factor    VIIa activity. J. Thromb. Haemost. 16:1817-1829 (2018).-   Fukudome K, et al. Identification, cloning, and regulation of a    novel endothelial cell protein C/activated protein C receptor. J.    Biol. Chem. 269:26486-26491 (1994).-   Ghosh S, et al. Endothelial cell protein C receptor acts as a    cellular receptor for factor VIIa on endothelium. J. Biol. Chem.    282:11849-11857 (2007).-   Grandoni J, et al. Kinetic analysis and binding studies of a new    recombinant human factor VIIa for treatment of haemophilia.    Haemophilia 23:300-308 (2017).-   Hedner U. Recombinant activated factor VII as a universal    haemostatic agent. Blood Coagul. Fibrinolysis. March; 9 Suppl    1:S147-152 (1998).-   Hoffman M, et al. Platelet binding and activity of a factor VIIa    variant with enhanced tissue factor independent activity. J. Thromb.    Haemost. 9:759-766 (2011).-   Jin J, et al. Factor VIIa's first epidermal growth factor-like    domain's role in catalytic activity. Biochemistry 38:1185-1192    (1999).-   Kaufman R J. Post-translational Modifications Required for    Coagulation Factor Secretion and Function. Thromb. Haemost.    79:1068-1079 (1998).-   Keshava S, et al. Factor VIIa interaction with EPCR modulates the    hemostatic effect of rFVIIa in hemophilia therapy: mode of its    action. Blood Adv. 1:1206-1214 (2017).-   Kisiel W. Human Plasma Protein C. J. Clin. Invest. 64:761-769    (1979).-   Kriegler T, et al. Measuring Endoplasmic Reticulum Signal Sequences    Translocation Efficiency Using the Xbp1 Arrest Peptide. Cell Chem.    Biol. 25:880-890 (2018).-   Lazarus R A, et al. Inhibitors of Tissue Factor-Factor VIIa for    Anticoagulant Therapy. Curr. Med. Chem. 11:2275-2290 (2004).-   Monroe D M, et al. Platelet activity of high-dose Factor VIIa is    independent of tissue factor. Br. J. Haematol. 99:542-547 (1997).-   Pan L C, et al. The propeptide of rat bone gamma-carboxyglutamic    acid protein shares homology with other vitamin K-dependent protein    precursors. Proc. Natl. Acad. Sci. USA 82:6109-6113 (1985).-   Stanley T B, et al. The Propeptides of the Vitamin K-dependent    Proteins Possess Different Affinities for the Vitamin K-dependent    Carboxylase. J. Biol. Chem. 274:16940-16944 (1999).-   Vavalle J P, et al. The effect of the REG2 Anticoagulation System on    thrombin generation kinetics: a pharmacodynamic and pharmacokinetic    first-in-human study. J. Thromb. Thrombolysis 38:275-284 (2014).-   von Bruhl M L, et al. Monocytes, neutrophils, and platelets    cooperate to initiate and propagate venous thrombosis in mice in    vivo. J. Exp. Med. 209:819-835 (2012).-   Yank V, et al. Systematic review: benefits and harms of in-hospital    use of recombinant factor VIIa for off-label indications. Ann.    Intern Med. 154:529-540 (2011).

The present invention is further described by the following claims.

1. A chimeric Protein C-Factor VII (PC-FVII) protein comprising a Gladomain of Protein C (PC), an EGF-1 domain of PC, an EGF-2 domain ofFactor VII (FVII), and a protease domain of FVII.
 2. The chimericPC-FVII protein according to claim 1, comprising (i) a light chaincomprising the Gla, EGF-1, and EGF-2 domains; and (ii) a heavy chaincomprising the protease domain.
 3. The chimeric PC-FVII proteinaccording to claim 2, wherein the light chain and the heavy chain arelinked by a disulfide bond.
 4. The chimeric PC-FVII protein according toclaim 1, wherein the Gla domain comprises the amino acid sequence setforth in SEQ ID NO:
 9. 5. The chimeric PC-FVII protein according toclaim 1, wherein the EGF-1 domain comprises the amino acid sequence setforth in SEQ ID NO:
 12. 6. The chimeric PC-FVII protein according toclaim 1, wherein the EGF-2 domain comprises the amino acid sequence setforth in SEQ ID NO:
 11. 7. The chimeric PC-FVII protein according toclaim 1, wherein the protease domain comprises the amino acid sequenceset forth in SEQ ID NO:
 14. 8. The chimeric PC-FVII protein according toclaim 1, wherein the protease domain comprises the amino acid sequenceset forth in SEQ ID NO:
 15. 9. The chimeric PC-FVII protein according toclaim 1, comprising the amino acid sequence set forth in SEQ ID NO: 6.10. The chimeric PC-FVII protein according to claim 1, comprising apropeptide sequence, wherein the propeptide sequence is capable ofbinding vitamin K-dependent γ-glutamyl carboxylase.
 11. The chimericPC-FVII protein according to claim 10, further comprising an endoplasmicreticulum translocalization signal peptide.
 12. The chimeric PC-FVIIprotein according to claim 11, comprising the amino acid sequence setforth in SEQ ID NO:
 5. 13. A composition comprising the chimeric PC-FVIIprotein according to claim
 1. 14. The composition according to claim 13,which is a pharmaceutical composition.
 15. A kit comprising the chimericPC-FVII protein according to claim
 1. 16. The kit according to claim 15,wherein the composition is contained in a pre-filled syringe.
 17. Anucleic acid encoding the chimeric PC-FVII protein according to claim 1.18. The nucleic acid according to claim 17, comprising the nucleotidesequence set forth in SEQ ID NO:
 7. 19. A method of activating Factor X(FX), the method comprising contacting FX with the chimeric PC-FVIIprotein according to claim 2, wherein the chimeric PC-FVII proteincleaves FX, thereby producing activated Factor X (FXa).
 20. The methodaccording to claim 19, which method is performed in the absence oftissue factor (TF).
 21. The method according to claim 19, wherein themethod is performed in blood or plasma.
 22. The method according toclaim 21, wherein contacting FX with the chimeric PC-FVII proteinresults in increased thrombin concentration in the blood or plasma,compared to contacting FX with wild-type or recombinant FVIIa.
 23. Achimeric PC-F VII protein according to claim 1 for use in activatingFactor X (FX).
 24. The use according to claim 23, wherein activating FXin blood or plasma results in increased thrombin concentration in theblood or plasma, compared to contacting FX with wild-type or recombinantFVIIa.